Discovery of diverse thyroid hormone receptor antagonists by high-throughput docking.

Treatment of hyperthyroidism, a common clinical condition that can have serious manifestations in the elderly, has remained essentially unchanged for >30 years. Directly antagonizing the effect of the thyroid hormone at the receptor level may be a significant improvement for the treatment of hyperthyroid patients. We built a computer model of the thyroid hormone receptor (TR) ligand-binding domain in its predicted antagonist-bound conformation and used a virtual screening algorithm to select 100 TR antagonist candidates out of a library of >250,000 compounds. We were able to obtain 75 of the compounds selected in silico and studied their ability to act as antagonists by using cultured cells that express TR. Fourteen of these compounds were found to antagonize the effect of T3 on TR with IC50s ranging from 1.5 to 30 microM. A small virtual library of compounds, derived from the highest affinity antagonist (1-850) that could be rapidly synthesized, was generated. A second round of virtual screening identified new compounds with predicted increased antagonist activity. These second generation compounds were synthesized, and their ability to act as TR antagonists was confirmed by transfection and receptor binding experiments. The extreme structural diversity of the antagonist compounds shows how receptor-based virtual screening can identify diverse chemistries that comply with the structural rules of TR antagonism.

Liver X receptors as insulin-mediating factors in fatty acid and cholesterol biosynthesis.

The nuclear receptor liver X receptor (LXR) alpha, an important regulator of cholesterol and bile acid metabolism, was analyzed after insulin stimulation in liver in vitro and in vivo. A time- and dose-dependent increase in LXRalpha steady-state mRNA level was seen after insulin stimulation of primary rat hepatocytes in culture. A maximal induction of 10-fold was obtained when hepatocytes were exposed to 400 nm insulin for 24 h. Cycloheximide, a potent inhibitor of protein synthesis, prevented induction of LXRalpha mRNA expression by insulin, indicating that the induction is dependent on de novo synthesis of proteins. Stabilization studies using actinomycin D indicated that insulin stimulation increased the half-life of LXRalpha transcripts in cultured primary hepatocytes. Complementary studies where rats and mice were injected with insulin induced LXRalpha mRNA levels and confirmed our in vitro studies. Furthermore, deletion of both the LXRalpha and LXRbeta genes (double knockout) in mice markedly suppressed insulin-mediated induction of an entire class of enzymes involved in both fatty acid and cholesterol metabolism. The discovery of insulin regulation of LXR in hepatic tissue as well as gene targeting studies in mice provide strong evidence that LXRs plays a central role not only in cholesterol homeostasis, but also in fatty acid metabolism. Furthermore, LXRs appear to be important insulin-mediating factors in regulation of lipogenesis.

Biological activity of mammalian transcriptional repressors.

Research on the regulation of transcription in mammals has focused in recent years mainly on the mechanism of transcriptional activation. However, transcriptional repression mediated by repressor proteins is a common regulatory mechanism in mammals and might play an important role in many biological processes. To understand the molecular mechanism of transcriptional repression, the activity of eight mammalian repressors or repressor domains was investigated using a set of model promoters in combination with two different transcriptional detection methods. The repressors studied were: REST, the thyroid hormone receptors alpha and beta, the zinc finger protein NK10 containing a 'krüppel-associated box' (KRAB), repressor domains derived from the proteins Egr-1, Oct2A and Dr1 and the repressor/activator protein YY1. Here we show that the repressor domains of REST, Egr-1, the thyroid hormone receptors alpha< and beta and NK10 were transferable to a heterologous DNA-binding domain and repressed transcription from proximal and distal positions. Moreover, these repressor domains also blocked the activity of a strong viral enhancer in a 'remote position'. Thus, these domains are 'general' transcriptional repressor domains. The 'krüppel-associated box' was the most powerful repressor domain tested. In contrast, the repressor domains derived from Oct2A and Dr1 were inactive when fused to a heterologous DNA-binding domain. The repressor domain of YY1 exhibited transcriptional repression activity only in one of the transcriptional assay systems. The recruitment of histone deacetylases to the proximity of the basal transcriptional apparatus was recently discussed as a mechanism for some mammalian transcriptional repressor proteins. Here we show here that histone deacetylase 2, targeted to the reporter gene via DNA-protein interaction, functions as a transcriptional repressor protein regardless of the location of its binding site within the transcription unit.

Vitamin D receptor displays DNA binding and transactivation as a heterodimer with the retinoid X receptor, but not with the thyroid hormone receptor.

The vitamin D receptor (VDR) is a transcription factor believed to function as a heterodimer with the retinoid X receptor (RXR). However, it was reported [Schräder et al., 1994] that, on putative vitamin D response elements (VDREs) within the rat 9k and mouse 28k calcium binding protein genes (rCaBP 9k and mCaBP 28k), VDR and thyroid hormone receptor (TR) form heterodimers that transactivate in response to both 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and triiodothyronine (T(3)). We, therefore, examined associations of these receptors on the putative rCaBP 9k and mCaBP 28k VDREs, as well as on established VDREs from the rat osteocalcin (rOC) and mouse osteopontin (mOP) genes, plus the thyroid hormone response element (TRE) from the rat myosin heavy chain (rMHC) gene. In gel mobility shift assays, we found no evidence for VDR-TR heterodimer interaction with any tested element. Further, employing these hormone response elements linked to reporter genes in transfected cells, VDR and TR mediated responses to their cognate ligands only from the rOC/mOP and rMHC elements, respectively, while the CaBP elements were unresponsive to any combination of ligand(s). Utilizing the rOC and mOP VDREs, two distinct repressive actions of TR on VDR-mediated signaling were demonstrated: a T(3)-independent action, presumably via direct TR-RXR competition for DNA binding, and a T(3)-dependent repression, likely by diversion of limiting RXR from VDR-RXR toward the formation of TR-RXR heterodimers. The relative importance of these two mechanisms differed in a response element-specific manner. These results may provide a partial explanation for the observed association between hyperthyroidism and bone demineralization/osteoporosis.

Mechanism of corepressor binding and release from nuclear hormone receptors.

The association of transcription corepressors SMRT and N-CoR with retinoid and thyroid receptors results in suppression of basal transcriptional activity. A key event in nuclear receptor signaling is the hormone-dependent release of corepressor and the recruitment of coactivator. Biochemical and structural studies have identified a universal motif in coactivator proteins that mediates association with receptor LBDs. We report here the identity of complementary acting signature motifs in SMRT and N-CoR that are sufficient for receptor binding and ligand-induced release. Interestingly, the motif contains a hydrophobic core (PhixxPhiPhi) similar to that found in NR coactivators. Surprisingly, mutations in the amino acids that directly participate in coactivator binding disrupt the corepressor association. These results indicate a direct mechanistic link between activation and repression via competition for a common or at least partially overlapping binding site.

Quantification of thyroid hormone receptor isoforms, 9-cis retinoic acid receptor gamma, and nuclear receptor co-repressor by reverse-transcriptase PCR in maturing and adult skeletal muscles of rat.

Quantitative competitive reverse-transcriptase polymerase chain reaction (qcRT-PCR) was established for determining absolute molecule numbers of the thyroid hormone receptor (T3R) isoforms T3Ralpha1, T3Ralpha2, T3Rbeta1, and the 9-cis retinoic acid receptor gamma (RXRgamma) in developing and adult fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles of rat. Expression levels of the nuclear receptor co-repressor (NCoR) were measured in the same muscles because responses to thyroid hormones during muscle maturation might not only depend on the expression levels of the various receptors but might also be modulated by changes in the expression of NCoR. The qcRT-PCR method was based on the addition of known amounts of homologous competitor RNAs to the reverse transcriptase (RT) reaction. We show that all nuclear receptors under study were expressed in fast and slow muscles. Transcript numbers of T3Rbeta1, which was the most abundant isoform, were higher in SOL than in EDL during all developmental stages. The mRNAs for T3Ralpha1, T3Ralpha2, RXRgamma and the NCoR displayed molecule numbers in similar ranges, but were differentially expressed. T3Ralpha1 mRNA increased in SOL during postnatal development, while T3Ralpha2 mRNA initially decreased, then increased to adult levels. Conversely, pronounced decreases were observed for T3Ralpha1 (10-fold) and T3Ralpha2 (28-fold) mRNAs in the EDL muscle during postnatal maturation. RXRgamma mRNA was 10-fold downregulated during EDL maturation, but unaltered in maturing SOL. NCoR transcript number displayed only minor changes in both muscles.

Nuclear receptors, nuclear-receptor factors, and nuclear-receptor-like orphans form a large paralog cluster in Homo sapiens.

We studied a human protein paralog cluster formed by 38 nonredundant sequences taken from the Swiss-Prot database and its supplement, TrEMBL. These sequences include nuclear receptors, nuclear-receptor factors and nuclear-receptor-like orphans. Working separately with both the central cysteine-rich DNA-binding domain and the carboxy-terminal ligand-binding domain, we performed multialignment analyses that included drawings of paralog trees. Our results show that the cluster is highly multibranched, with considerable differences in the amino acid sequence in the ligand-binding domain (LBD), and 17 proximal subbranches which are identifiable and fully coincident when independent trees from both domains are compared. We identified the six recently proposed subfamilies as groups of neighboring clusters in the LBD paralog tree. We found similarities of 80%-100% for the N-terminal transactivation domain among mammalian ortholog receptors, as well as some paralog resemblances within diverse subbranches. Our studies suggest that during the evolutionary process, the three domains were assembled in a modular fashion with a nonshuffled modular fusion of the LBD. We used the EMBL server PredictProtein to make secondary-structure predictions for all 38 LBD subsequences. Amino acid residues in the multialigned homologous domains--taking the beginning of helix H3 of the human retinoic acid receptor-gamma as the initial point of reference--were substituted with H or E, which identify residues predicted to be helical or extended, respectively. The result was a secondary structure multialignment with the surprising feature that the prediction follows a canonical pattern of alignable alpha-helices with some short extended elements in between, despite the fact that a number of subsequences resemble each other by less than 25% in terms of the similarity index. We also identified the presence of a binary patterning in all of the predicted helices that were conserved throughout the 38-sequence sample. Our results fit well with a recently proposed evolutionary model that combines protein secondary structure and amino acid replacement. We propose a new hypothesis for molecular evolution, in which chaperones--acting as an endogenous cellular device for selection--play a crucial role in preserving protein secondary structure.

Cloning and expression of a brain-derived TSH receptor.

Several hormones not only regulate the activity of endocrine cells and non-endocrine tissues but also serve as neuronal transmitters or modulators of neuronal activity. Accordingly, the expression and physiological significance of hormonal receptors in the central nervous system (CNS) could be demonstrated for a whole set of hormones (e.g. hCG/LH, GH, T3, CRF, TRH). The G-protein coupled TSH receptor is densely expressed in the thyroid gland and mediates the production and secretion of thyroid hormones. Not all TSH effects, especially in neurological and psychiatric disease states, can readily be explained by the action of the hormone on the thyroid gland and/or TRH levels. Therefore, it has been suggested that TSH might exert its effects directly in the CNS, although no direct proof for a TSH receptor in the human brain has been provided yet. Here we describe the cloning of a TSH receptor from an ovine hypothalamic cDNA library that is similar to thyroid derived cDNA clones. The comparison of amino acid sequences indicates that several protein domains important for the function and activity of the receptor are highly conserved. RT-PCR and RNA protection assay demonstrated that the TSH receptor mRNA is widely expressed throughout the ovine brain. The expression of a TSH receptor in the CNS indicates that TSH is not only a hormonal messenger for the thyroid gland but can also act directly in the brain. Further studies should focus on the physiological role of TSH in the CNS and the regulation of TSH receptor expression in the mammalian brain.

Genomic structure, promoter identification, and chromosomal mapping of a mouse nuclear orphan receptor expressed in embryos and adult testes.

We have isolated and characterized overlapping genomic clones containing the complete transcribed region of a newly isolated mouse cDNA encoding an orphan receptor expressed specifically in midgestation embryos and adult testis. This gene spans a distance of more than 50 kb and is organized into 13 exons. The transcription initiation site is located at the 158th nucleotide upstream from the translation initiation codon. All the exon/intron junction sequences follow the GT/AG rule. Based upon Northern blot analysis and the size of the transcribed region of the gene, its transcript was determined to be approximately 2.5 kb. Within approximately 500 bp upstream from the transcription initiation site, several immune response regulatory elements were identified but no TATA box was located. This gene was mapped to the distal region of mouse chromosome 10 and its locus has been designated Tr2-11. Immunohistochemical studies show that the Tr2-11 protein is present mainly in advanced germ cell populations of mature testes and that Tr2-11 gene expression is dramatically decreased in vitamin A-depleted animals.

NOT, a human immediate-early response gene closely related to the steroid/thyroid hormone receptor NAK1/TR3.

By analyzing the early genetic response of human T cells following mitogenic activation we have identified NOT, a member of the steroid/thyroid hormone family of receptors. NOT has all structural features of steroid/thyroid hormone receptors (C2C2 zinc-finger domain, ligand binding domain), but is rapidly and only very transiently expressed after cell activation, which is clearly at variance with classical steroid receptors such as glucocorticoid or estrogen receptors. NOT gene induction is independent of de novo protein synthesis, defining NOT as an immediate-early response gene. Short-lived NOT mRNA (4.2 kilobases) expression could be observed in vitro in a greater number of tissue types following activation by a variety of distinct stimuli. In vivo, NOT mRNA expression was detected exclusively in the brain, where a very strong signal was observed. By immunoblot analysis of human T cell lysates with NOT specific antisera two activation-dependent protein bands (66 and 59 kilodaltons) could be detected. NOT gene was localized to human chromosome 2q22-q23. Sequence comparison revealed that NOT is the human homolog of the murine NURR1 and rat RNR-1. Moreover NOT is closely related to NAK1/TR3, a previously identified human orphan steroid receptor. Several lines of evidence indicate that NOT and NAK1/TR3 form a distinct and exclusive subgroup of orphan steroid receptors, whose expression characteristics in vitro and in vivo resemble the expression of nonsteroid immediate-early transcription factors such as jun and fos. NOT and NAK1/TR3 thus may function as general coactivators of gene transcription rather than participate in the induction of specific target genes, as is the case with classical steroid receptors.

Expression of nuclear hormone receptors within the rat hippocampus: identification of novel orphan receptors.

Within the hippocampus, stimulus-transcriptional coupling plays an important role in post-seizure neuronal adaptation, post-ischemic cell death and the induction of long-term potentiation. To identify additional mediators of hippocampal transcriptional responses a targeted approach was developed and used to characterize the spectrum of nuclear hormone receptors expressed within this brain region. cDNAs encoding the DNA-binding domains of six different members of the nuclear hormone receptor superfamily were isolated. A majority were identical or closely related to receptors known to be expressed within the hippocampus. Two additional isolates, HZF-2 and HZF-3, encode the DNA-binding domain of novel members of the nuclear hormone receptor superfamily.

Molecular cloning, cDNA analysis, and localization of a monomer of the N-acetylglucosamine-specific receptor of the thyroid, NAGR1, to chromosome 19p13.3-13.2.

We have proposed that the GlcNAc thyroid receptor triggers selective recycling of immature GlcNAc-bearing thyroglobulin molecules through the Golgi back to the apical membrane for further processing until maturation is achieved. This process, which we call "receptor-mediated exocytosis," prevents lysosomal degradation of thyroid prohormones. In the present study, we report cloning of the cDNA encoding the (or one of the) monomer(s) constituting the human GlcNAc thyroid receptor. This novel gene, called NAGR1, was assigned by in situ hybridization to subbands p13.3-p13.2 of chromosome 19. Northern blot analysis showed that the mRNA encoding NAGR1 was present as a single transcript of 2.1 kb in the thyroid, but not in the heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. The deduced amino acid sequence comprised a 51-kDa type I membrane protein with a single spanning region and a short intracytoplasmic domain. Sequence analysis showed that NAGR1 is a glycine-, tryptophan-, and methionine-rich protein with no cysteine residues or glycosylation site. No sequence homology with any known cDNA or protein was noted. The extracellular domain is composed of 420 amino acids and contains a region of 204 residues showing 15 repeats of 4 amino acids, each 1 having an acidic amino acid presumably involved in calcium coordination. The intracellular domain contained what appeared to be a tyrosine internalization signal. The usefulness of this clone in glycobiology, cell biology, and thyroid pathology studies is discussed.

Physiology of the steroid-thyroid hormone nuclear receptor superfamily.

Glucocorticoids, other steroid hormones, thyroid hormones and vitamin-derived hormones (including retinoids) all exert their effects by the regulation of hormone-responsive target genes within the cell nucleus. These hormones bind to a series of specific nuclear receptor proteins that function as hormone-inducible transcription factors. The receptors are structurally homologous, are related to the avian erythroblastosis oncogene v-erbA, and exhibit remarkable evolutionary conservation. Together they form the steroid-thyroid hormone nuclear receptor superfamily. This chapter describes the structure and functions of the various family members and highlights the differences and similarities that occur between individual receptor proteins. Type I receptors, which include glucocorticoid receptor and other steroid receptor proteins, interact as homodimers with target sequences of DNA containing two receptor binding sites arranged as a palindrome. Type II receptors, which include receptors for retinoids, thyroid hormone and vitamin D3, bind as heterodimers (or homodimers) to DNA sequences in which two or more receptor-binding sites are arranged as a direct repeat or as other more complex configurations. The complexity of both receptor-DNA and receptor-receptor interactions predicts the potential for considerable cross-talk between various hormone-activated pathways. Thus, the specificity of hormone action and its regulation is discussed in relation to the structural and functional characteristics of the receptors and their molecular mechanisms of action. Finally, potential sites of regulation of hormone action, from circulating hormone levels in the periphery to their delivery to the cell and final site of action in the nucleus, are highlighted to provide a perspective for the following chapters in this volume and to indicate their clinical significance.

Formation of retinoid X receptor homodimers leads to repression of T3 response: hormonal cross talk by ligand-induced squelching.

Thyroid hormone receptors (TRs) form heterodimers with retinoid X receptors (RXRs). Heterodimerization is required for efficient TR DNA binding to most response elements and transcriptional activation by thyroid hormone. RXRs also function as auxiliary proteins for several other receptors. In addition, RXR alpha can be induced by specific ligands to form homodimers. Here we report that RXR-specific retinoids that induce RXR homodimers are effective repressors of the T3 response. We provide evidence that this repression by RXR-specific ligands occurs by sequestering of RXR from TR-RXR heterodimers into RXR homodimers. This ligand-induced squelching may represent an important mechanism by which RXR-specific retinoids and 9-cis retinoic acid mediate hormonal cross talk among a subfamily of nuclear receptors activated by structurally unrelated ligands.

Region-specific anti-thyroid hormone receptor (TR) antibodies detect changes in TR structure due to ligand-binding and dimerization.

There are multiple factors that potentially can induce structural changes in DNA-bound thyroid hormone receptors (TRs) including protein-protein interactions, ligand-binding to TRs, and the thyroid hormone response element (TRE) sequence. We used a battery of anti-TR antibodies that recognize the amino-terminal, hinge, or carboxy-terminal regions of TRs to study changes in the epitope regions of in vitro translated TRs in electrophoretic mobility shift assays. We found that the carboxy-terminal and hinge region antibodies recognized TR homodimers but not TR/T3-receptor auxiliary protein or TR/retinoid X receptor heterodimers. The amino-terminal antibodies detected conformational changes due to ligand binding. In contrast, each antibody recognized TR complexes bound to TREs containing half-sites arranged in three different orientations. These results suggest that dimerization with nuclear proteins and ligand-binding, rather than the orientation of TRE half-sites, cause changes in several TR subregions.